The present invention relates to a biosensor facilitating simple and prompt quantitation of a specific component contained in a sample with high accuracy.
Conventionally, various biosensors have been proposed to make simple quantitation of a specific component contained in a sample without the need of dilution or agitation of a sample solution. The following is one example of such biosensor (Japanese Laid-Open Patent Publication Hei 2-062952).
The biosensor disclosed in this reference is produced by the steps of forming an electrode system including a working electrode and a counter electrode on an electrically insulating base plate using a screen printing method or the like and subsequently forming immediately above this electrode system an enzyme reaction layer including a hydrophilic polymer, an oxidoreductase and an electron acceptor. The enzyme reaction layer may further contain a buffer if occasion demands.
When the biosensor thus produced is added with a drop of a sample solution containing a substrate over the enzyme reaction layer, dissolution of the enzyme reaction layer in the sample solution will occur, which triggers a reaction between the enzyme and the substrate thereby causing reduction of the electron acceptor. Upon completion of enzyme reaction, the reduced electron acceptor is oxidized electrochemically. From the oxidation current value measured during this oxidizing step, the concentration of the substrate in the sample solution can be quantitated.
The biosensor as described above permits measurements of various materials in principle if a suitable enzyme corresponding to the substrate of a target material is selected. Enzymes, which normally contain protein as their main component, are often purified before their use in a sensor. Depending on the condition of purification, separation of metallic ion, which is a major active constituent of an enzyme, may occur, causing a change of the cubic structure of the enzyme. As a result, the enzyme may be changed in its substrate specificity or lose its activity.
Moreover, degeneration of the enzyme may occur during preservation of the sensor including the enzyme due to absorption of water in the atmosphere. Therefore, in determining the amount of enzyme to be contained in the reaction layer, the amount of loss due to possible degeneration during preservation must be taken into account.
On the other hand, in such a biosensor whose enzyme reaction is dependent on the amount of the enzyme contained in the reaction layer, responsive current values measured are not proportional to the concentration of the substrate. Therefore, it is required for the reaction layer to contain a sufficient amount of enzyme which is enough to effect a reaction with the target substrate in the sample solution.
As such, at production of a sensor, it is necessary to include in the reaction layer considerably excess amounts of enzyme than the amount of the substrate which is anticipated to exist in the sample solution. Accordingly, the required amount of enzyme per sensor is assumed to become great, which results in a significant increase of the cost per sensor.